Abstract

Using a rabbit model of tuberculous meningitis, we evaluated the protective efficacy of vaccination with the recombinant polyprotein Mtb72F, which is formulated in two alternative adjuvants, AS02A and AS01B, and compared this to vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) alone or as a BCG prime/Mtb72F-boost regimen. Vaccination with Mtb72F formulated in AS02A (Mtb72F+AS02A) or Mtb72F formulated in AS01B (Mtb72F+AS01B) was protective against central nervous system (CNS) challenge with Mycobacterium tuberculosis H37Rv to an extent comparable to that of vaccination with BCG. Similar accelerated clearances of bacilli from the cerebrospinal fluid, reduced leukocytosis, and less pathology of the brain and lungs were noted. Weight loss of infected rabbits was less extensive for Mtb72F+AS02A-vaccinated rabbits. In addition, protection against M. tuberculosis H37Rv CNS infection afforded by BCG/Mtb72F in a prime-boost strategy was similar to that by BCG alone. Interestingly, Mtb72F+AS01B induced better protection against leukocytosis and weight loss, suggesting that the polyprotein in this adjuvant may boost immunity without exacerbating inflammation in previously BCG-vaccinated individuals.

Effect of AS01B and AS02A adjuvant on Mtb72F-induced protection against CNS M. tuberculosis infection and inflammation (n = 4 rabbits/group). Values are means. Error bars indicate standard errors of the means. (A) Number of CFU of M. tuberculosis in the CSF for control nonvaccinated rabbits (black bars), BCG-vaccinated rabbits (white bars), rabbits vaccinated with Mtb72F+AS02A (light gray bars), and rabbits vaccinated with Mtb72F+AS01B (dark gray bars). Bacillary clearance was significantly accelerated in all vaccinated groups compared to that of the controls (P was 0.0004 for BCG, 0.0007 for Mtb72F+AS02A, and 0.008 for Mtb72F+AS01B). (B) WBC density in CSF for control nonvaccinated rabbits (black squares), rabbits vaccinated with BCG (open squares), rabbits vaccinated with Mtb72F+AS02A (open triangles), and rabbits vaccinated with Mtb72F+AS01B (×). Leukocyte counts were significantly reduced in all vaccinated groups compared to those of nonvaccinated rabbits (P was <0.0001 for BCG, 0.0002 for Mtb72F+AS02A, and 0.0003 for Mtb72F+AS01B), with no significant difference among the vaccinated groups. (C) Body weight change (%) for control nonvaccinated rabbits (black squares), rabbits vaccinated with BCG (open squares), rabbits vaccinated with Mtb72F+AS02A (open triangles), and rabbits vaccinated with Mtb72F+AS01B (×). Rabbits vaccinated with Mtb72F+AS02A showed the least change in the body weight compared to that of the control nonvaccinated animals (P = 0.0001).

Effect of Mtb72F+AS02A vaccination of rabbits on bacillary load in the CSF and in tissues (n = 8 rabbits/group). Values are means. Error bars indicate standard errors of the means. (A) Numbers of CFU of M. tuberculosis (CFU/ml) in the CSF of control nonvaccinated rabbits (black bars), BCG-vaccinated rabbits (white bars), and rabbits vaccinated with Mtb72F in AS02A (gray bars). Clearance of bacilli from the CSF was significant for both vaccinated groups (P < 0.0001) compared to that for the nonvaccinated control animals. (B) CFU in the brains, lungs, and livers 8 weeks postinfection for control nonvaccinated rabbits (black bars), BCG-vaccinated rabbits (white bars), and rabbits vaccinated with Mtb72F in AS02A (gray bars). Significantly smaller amounts of CFU were observed in the brains of rabbits from both vaccination groups (P was 0.03 for BCG and 0.02 for Mtb72F+AS02A) and in the lungs of BCG-vaccinated rabbits (P = 0.03). No dissemination of bacilli to the livers was seen in any of the vaccinated rabbits. No significant difference was noted between BCG and Mtb72F+AS02A.

Effect of vaccination with Mtb72F+AS02A on the CSF inflammatory response, body weight, and pathology in brain and lungs (n = 8 rabbits/group). Values are means. Error bars indicate standard errors of the means. (A) WBC density in CSF for control nonvaccinated rabbits (black squares), rabbits vaccinated with BCG (open squares), and rabbits vaccinated with Mtb72F+AS02A (gray squares). Significantly reduced leukocytosis was observed in both vaccinated groups (P was 0.0004 for BCG and <0.0001 for Mtb72F+AS02A). (B) Body weight change (%) for control nonvaccinated rabbits (black squares), rabbits vaccinated with BCG (open squares), rabbits vaccinated with Mtb72F in AS02A (gray squares). Rabbits that were vaccinated with Mtb72F+AS02A showed significantly higher body weights compared to those of the control animals (P = 0.0001) and compared to that of the BCG-vaccinated group (P = 0.03). (C) Severity of pathology in the brain and lungs for control nonvaccinated rabbits (black bars), BCG-vaccinated rabbits (white bars), and rabbits that were vaccinated with Mtb72F+AS02A (gray bars). BCG-vaccinated rabbits showed significantly less pathology in the brain (P = 0.001). Pathology in the lung was significantly less in both vaccinated groups (P was <0.001 for BCG and 0.008 for Mtb72F+AS02A).

Histopathology of the meninges and brain of rabbits infected intrathecally with M. tuberculosis H37Rv (8 weeks postinfection). (A and a) Control nonvaccinated rabbit. (A) Moderate inflammation of the meninges with high numbers of inflammatory cells, lymphocytes, and macrophages (mac) and partial necrosis (nec). (a) Vasculitis is seen not only in the meninges but also in the brain parenchyma. (B and b) Mild focal inflammation within the meninges with no evidence of vasculitis for rabbit vaccinated with BCG. (C and c) Moderate meningitis associated with perivascular inflammation for a rabbit vaccinated with Mtb72F+AS02A. Note the lymphocytes infiltrating the vessel wall (C). No signs of vasculitis were observed in the brain parenchyma (c). (D and d) Rabbit vaccinated with BCG and boosted with Mtb72F+AS01B. Very mild focal inflammation is seen in the meninges with few lymphocytes within the subarachnoid space (arrows), and no signs of vasculitis are seen. Blood vessels are labeled V. Sections are stained with hematoxylin and eosin. Magnification, ×40.

Histopathology of the lung of rabbits infected intrathecally with M. tuberculosis H37Rv (8 weeks postinfection). (A and a) Control nonvaccinated rabbit. Big confluent lung granulomas (G) are shown outlined by the demarcation line, occupying most of the lung parenchyma, with central necrosis (nec). Large numbers of foamy nondifferentiated macrophages (mac) and lymphocytes (ly) are seen. (B and b) Rabbit vaccinated with BCG. No granulomas were seen, but increased accumulation of mononuclear leukocytes in the alveolar spaces was noted. (C and c) Rabbit vaccinated with Mtb72F+AS02A. Very few small and well-organized granulomas were observed. Note the mature activated epitheloid macrophages (mac) and numerous lymphocytes (ly). (D and d) Rabbit vaccinated with BCG and boosted with Mtb72F+AS02A. No granulomas were observed, only increased cellularity in the alveolar walls. Alveolar spaces are labeled a. Sections are stained with hematoxylin and eosin. Magnification, ×10 for panels A, B, C, and D and ×40 for panels a, b, c, and d.

Proliferative activity of rabbit peripheral blood CD4+ lymphocytes, following incubation with BCG for 6 days, as measured by BrdU incorporation. Results for control nonvaccinated rabbits (control), rabbits vaccinated with BCG only (BCG), and rabbits vaccinated with BCG and boosted with Mtb72F+AS01B are shown. Values are means of four rabbits/group. Error bars indicate standard errors of the means.